Not Applicable.
This invention relates to the assessment of invasive potential of biological cells. More specifically, this invention is directed to the testing of tumor cells for their ability to invade tissues and cause metastases.
Metastasis, i.e. the spread of tumor cells from a primary tumor to distant organs, is the major cause of death for most cancer patients. Metastatic process consists of several essential events including escape of tumor cells from the bulk tumor, survival of tumor cells in the circulation, and migration of tumor cells to distance sites where conditions are suitable for growth.
Cell invasion of basement membrane is a crucial step in the multiple steps of tumor metastasis and is pathologically considered the hallmark of a malignant tumor. Basement membranes are thin continuous layers composed of a dense meshwork of laminin, collagen, glycoprotein, proteoglycan and other components. Basement membranes normally underlie epithelia and form barriers that block the passage of cells and macromolecules. However, most malignant tumor cells have acquired ability to invade and penetrate basement membrane barriers. The process of cell invasion includes: one or more tumor cells attach to basement membrane, secret enzymes to degrade adjacent basement membrane, pass through the basement membrane barrier, and migrate into adjacent tissue.
A variety of in vitro assays have been devised for evaluating the ability of tumor cells to penetrate the basement membrane. This is done by methods known in the prior art. Isolated natural basement membrane, submucosa-derived matrix, and basement membrane matrix derived from animal or human origin have been employed for cell invasiveness assay(Russo, R et al., U.S. Pat. No. 4,446,234 (1984); Kleiman, H K et al., U.S. Pat. No. 4,829,000(1989); Kleiman, H K et al., U.S. Pat. No. 5,158,874(1992); Livant, D L. U.S. Pat. No. 5,840,514(1998); Badlak, S F et al., U.S. Pat. No. 6,087,157(2000)). For example, the basement membrane matrix can be applied directly onto a culture plate or Petri dish to form a gel-like substrate for cell growth and invasion. The tested invasive tumor cells are able to degrade the substrate and migrate toward the bottom of the plate. Invaded tumor cells could be observed at the bottom plane of the plate through a microscope. Such thick gel method requires long experimental period and is unsatisfied for quantitative purpose. At the present time conventional or modified blind well Boyden chamber assays are most widely used for quantitative analysis of cell invasiveness.
Essentially, the conventional blind well Boyden chamber assay procedure involves placing a suspension of tumor cells and a chemical agent (chemoattractant) in two separate chambers. The two chambers are separated by a membrane filter (such as polycarbonate membrane), which is coated with a thin layer of extracellular matrix (such as Matrigel(copyright), Becton Dickinson, Bedford, Mass.). The tested tumor cells in one chamber migrate through the membrane filter into another chamber in response to chemotactic stimuli. After a predetermined period of time, the membrane filter is removed and cells on the filter surface closest to the chamber containing the cell suspension are removed. The remaining cells on the underside of the filter (i.e. the side of the filter closest to the chamber containing the chemoattractant) are then fixed and stained. Using a high power microscope, the filter is examined. The number of cells appearing on the underside of the filter is counted. This type of assay is usually referred as chemotaxis assay or chemoinvasion assay.
However, The application of blind well Boyden chamber assay involves removing the sample membranes with attached cells from the chambers after incubation. Such manipulation and following manipulations in staining process are prone to cell loss and make the cell counting inaccurate. Moreover, Invasive cells passed through the basement membrane into the lower chamber consist of two portions: one attached to the lower surface of the membrane and the other fell down at the bottom of the lower chamber. Although the cells fell down at the bottom of the lower chamber present very invasive potential, this portion of cells is usually discarded in the assay since additional procedures and time be needed to collect them. Finally, The conventional blind well Boyden chamber assay requires several preparatory steps including coating membrane, filling into lower chamber with medium with chemoattractant, assembling the upper chamber and filling into the upper chamber with cell suspension. These successive manipulations increase the interval between different samples and complicate the assay procedure.
There are several modifications of the blind well Boyden chamber applications. Some test instruments or kits are commercially available. These include: (1)transwell plate with polycarbonate membrane filter (Costar Scientific, Cambridge, Mass.), (2)multiwell plate with inserts (Becton Dickinson, Bedford, Mass.), and (3)ECM Invasion chamber including multiwell culture plate and inserts (Chemicon International, Temecula, Calif.). Although the membranes of the inserts or transwell plates are pre-coated with extracellular matrix substrate to facilitate preparatory procedure, those applications do not overcome the major disadvantages which affect the accuracy of the experiment.
There is a great need in developing a simple and more reliable device and method for evaluating cellular invasive potential of tumor cells.
The present invention is directed to the use of a simple apparatus with a single or multi test sites for cell invasiveness analysis which requires minimum manipulation. Specifically, various procedures such as cell culturing, separating, staining, and counting invaded cells of a test sample may be performed on the same plate of the apparatus to speed up testing and to minimize the cell loss.
The major components of the apparatus comprise a supporting plate, a microporous membrane, an intermediate layer of extracellular matrix deposited between the membrane and the plate, a top layer of the extracellular matrix coated on the surface of the membrane, and a bonding element to adhere periphery of the membrane to the plate. The optimal thickness of the top layer of the extracellular matrix, the optimal thickness of the intermediate layer of the extracellular matrix between the plate and the membrane, and the optimal pore size of the membrane are selected such that the cells under study can not pass through the pores directly. In order to pass through the pores of the membrane, the tested cells must: (a) attach, digest and absorb the coated extracellular matrix to access the membrane; (b) deform and migrate through the pores of the membrane; (c) digest and absorb the extracellular matrix deposited between the membrane and the plate; and (d) migrate into the intermediate layer between the membrane and the plate. These steps mimic the process of tumor cell invasion of basement membrane in metastasis in vivo.
The present invention provides a novel in vitro assay method for quantitative analysis of cell invasiveness. With the use of the test apparatus in accordance with the present invention, the invaded cells, without the effect of chemoattractant, pass through the membrane pores and enter the intermediate layer of the extracellular matrix between the membrane and the plate. Cells that fail to invade will remain on the surface of the membrane and can be excluded during experimental process.
A variety of assay formats are contemplated for testing the invasive potential of tumor cells. In one preferred embodiment, a semisolid tumor cell suspension (as a bulk tumor) is placed on the test site of the apparatus. Thereafter, the invasiveness of tumor cells in contact with extracellular matrix-coated membrane is assessed. Alternatively, a portion of a patient""s tumor is placed on the test site of the apparatus and the metastatic feature of the tumor tissue is assessed. In another preferred embodiment, a single cell suspension is placed on the test site of the apparatus, and the invasiveness of tumor cells is assessed.
Since all procedures of the cell invasiveness assay are performed on the same plate of the apparatus and all invaded cells are confined to the intermediate layer between the membrane and the plate in all manipulation steps, there are a number of advantages:
(a) there are no risks of cell loss during manipulations;
(b) all invaded cells may be counted;
(c) the invaded cells are confined near the plane that is suitable for microscopy so that the process of cell invasion can be microscopically examined at desired timepoints during the experiment period.
(d) there are no time-consuming preparatory manipulations for coating membrane and assembly of the two chambers.
It is a first object of the present invention to provide a simple apparatus and method which integrates and simplifies experimental procedure for measuring intrinsic invasiveness potential of cells, especially for malignant tumor cells.
It is a second object of the present invention to provide a method to evaluate the metastatic feature of malignant tumor specimen in vitro.
It is a third object of the present invention to provide an apparatus in which cells can be microscopically examined during experimental period.
It is a fourth object of the present invention to provide an apparatus that is inexpensive and disposable.
It is the fifth object of the present invention to provide an apparatus for the measurement of cell invasiveness using common laboratory equipment.
These and other objects and advantages of the present invention are achieved as described in the detailed description of the invention, the appended drawings and the attached claims.